Summary Approximately 101,000 people are waitlisted for a kidney transplant in the United States, while only about 17,000 kidney transplants took place in 2013. This shortage of transplantable kidneys can be overcome by differentiating kidneys from patient derived induced pluripotent-stem cells to improve survival rates of end stage renal disease (ESRD) patients. Since kidneys develop from the ureteric bud (UB) and metanephric mesenchyme (MM), we need to make the different cell types that differentiate from the UB and MM to engineer kidneys in culture. Our goal is to understand the molecular mechanisms regulating kidney collecting duct differentiation into principal cells (PCs) and intercalated cells (ICs). This knowledge will be used to establish methods to convert UB cells into PCs and ICs in culture. We hypothesize that PC promoting signals (Signal-P) co-operate with Notch to activate Elf5 expression and other PC lineage transcription factors (PCFs) to promote PC differentiation (Fig.1). Ectopic expression of activated Notch1 turns on Hes1 and Elf5 among other candidate PCFs to induce precocious PC differentiation in developing CDs in vivo, but is unable to turn on Elf5 or other PC specific genes in cultured immature UB cells. Notch signaling suppresses Foxi1 (an essential IC factor), other candidate IC transcription factors (ICFs), and components of candidate signaling pathways (Signal-I) that promote IC differentiation. In Aim1 the early regulators of collecting duct differentiation that turn on Elf5, an early PC marker, and Foxi1, an early intercalated cell (IC) marker, will be determined. Aim2 focuses on determining intermediate regulators of PC and IC differentiation by examining the contribution of Foxi1, Elf5 and Hes1 to collecting duct differentiation using genetically modified mice, UB cells, and mature PC and IC lines. In Aim3 we will determine how mature PCs are maintained. We have evidence implicating Notch signaling in maintenance of mature PC cell state. This will be verified by conditional inactivation of Notch signaling in adult CDs and the mechanisms of Notch mediated PC maintenance will be determined. A mouse model of acquired Nephrogenic Diabetes Insipidus (aNDI) in which mature PCs are reduced, potentially due to cell fate switching, will be used to determine which CD differentiation factors are involved in PC maintenance. By lineage tracing we will definitively determine whether PC to IC fate change occurs during aNDI. These studies are not merely incremental but provide a transformative step in our understanding of collecting duct development and maintenance.